Covalent modification by isoprenoid lipids (prenylation) is increasingly being recognized as an important component in the localization and activity of many proteins involved in signal transduction pathways. The discovery that the members of the ras family of oncogene products were modified by the farnesyl (15-carbon) prenyl group, and that prenylation was required for the ras proteins to express their transforming potential, has revealed a previously unrecognized mechanism for regulation of cell growth. Furthermore, a number of ras-related proteins, members of the so- called "small G protein" family, and the gamma subunits of the heterotrimeric G proteins are also now known to be prenylated - most of these proteins contain the 20-carbon geranylgeranyl group. In fact, the geranylgeranyl group appears to be the predominant prenyl group in mammalian protein modification. Geranylgeranylated proteins have been implicated in the regulation of a variety of cellular processes, including transmembrane signaling, intracellular membrane trafficking, and secretion, and a variety of studies have highlighted the importance of this prenyl modification in these regulatory pathways. The long term goal of this research project is to understand the molecular mechanisms of protein prenylation and the role that these modifications play in the targeting and biological activity of modified proteins. In particular, the emphasis will be on the 20-carbon prenyl modification, geranylgeranyl. The specific Aims of this proposal are: l) Determination of the primary structure of the protein geranylgeranyltransferase (PGGT), the enzyme responsible for the geranylgeranyl modification of a number of key proteins involved in cellular signaling; 2) Development of cDNA expression systems for mammalian PGGT; and 3) Initiation of structure/function analysis of mammalian PGGT. The methodologies (both biochemical and molecular biological) to be employed in these studies are well-established. Accomplishment of these Aims will serve to define systems that will greatly facilitate future investigations into protein prenylation, and the role of this process in control of cellular signaling events and oncogenesis.